CN114958621A - Method for culturing eurotium cristatum by using different tea water - Google Patents
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Abstract
The invention discloses a method for culturing eurotium cristatum by using different tea water, belonging to the technical field of microbial fermentation engineering. The method adopts microorganism separation culture technology and microorganism deep liquid fermentation technology, and prepares Eurotium cristatum strain by taking different tea water as fermentation culture medium. Firstly, drying loose tea of different varieties at high temperature, crushing and sieving to obtain loose tea powder, taking different tea water obtained by steaming and boiling the loose tea powder at high temperature as a liquid fermentation culture medium of eurotium cristatum, and then inoculating eurotium cristatum spore suspension for submerged fermentation. The method is simple and easy to operate, convenient to control and suitable for industrial production, and the eurotium cristatum mycelium pellets obtained by the method can also be put into the fields of medical care, food improvement and the like.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation engineering, and particularly relates to a method for culturing eurotium cristatum by using different tea water.
Background
Eurotium cristatum is commonly called as eurotium cristatum, is aerobic, drying-resistant and extremely strong in vitality, can grow under the condition that the pH value is 3-6, the optimal growth pH value is 5, and the optimal growth temperature is 28-30 ℃. Under different growth environments, different reproduction modes can be generated, namely sexual reproduction and asexual reproduction, wherein the sexual reproduction is realized in hypertonic condition, the sexual reproduction is realized in hypotonic condition, and the two reproduction modes are different greatly. The golden flower fungus is a golden yellow cyst shell generated in the Fuzhuan tea through natural 'blooming' of the Fuzhuan tea, is a dominant flora in the Fuzhuan tea processing process, and the quality and the quantity of the golden flower fungus are important indexes for evaluating the quality of the Fuzhuan tea.
At present, the production of dominant bacteria, namely eurotium cristatum, in the Fuzhuan tea is realized mainly by controlling the environmental conditions of temperature and humidity and naturally generating a large amount of golden yellow cyst closure shells, but the method has a single production means, the produced eurotium cristatum is less, and the quality of the produced Fuzhuan tea is uncontrollable. In addition, although fermentation of tea soup using eurotium cristatum has been reported, improvement of color, flavor and taste of tea soup is only achieved by using eurotium cristatum, and liquid fermentation of eurotium cristatum is not achieved by using tea soup, and thus improvement of quality of fu brick tea by increasing the number of eurotium cristatum in tea water has not been reported.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for culturing eurotium cristatum by using different tea water, and solves the problems of single means and uncontrollable quality in the production of eurotium cristatum by natural 'flowering'.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses a method for culturing eurotium cristatum by using different tea water, which comprises the steps of diluting and inoculating eurotium cristatum suspension on a solid tea soup culture medium, and culturing for 8-10 days at 28-30 ℃; the solid tea soup culture medium is prepared by adding agar powder into different tea water according to the proportion of 2 percent and sterilizing at high temperature under the condition of natural PH value;
or inoculating the eurotium cristatum spore suspension into a liquid tea soup culture medium with the sample loading of 40% v/v according to the inoculation amount of 7% v/v, and culturing at 28 ℃ and 150r/min for 6 d; the liquid tea soup culture medium is prepared by sterilizing different tea water at natural pH for 20min at high temperature.
Preferably, the preparation method of the different tea water comprises the following steps: drying, crushing and sieving different kinds of loose tea to obtain loose tea powder, and steaming and boiling the loose tea powder and distilled water at high temperature and filtering to obtain different tea water.
Further preferably, the different loose teas include raw dark green tea, yellow tea, oolong tea, black tea and green tea.
Preferably, the drying is drying at 40-60 ℃.
Preferably, the powdered tea is mixed with distilled water in a ratio of 1g to 100 mL.
Preferably, the high-temperature cooking is cooking at 60-90 ℃.
Preferably, the crushing and sieving is to pass through a 80-mesh sieve after crushing by a crusher with the power of 1000W.
Preferably, the preparation method of the eurotium cristatum suspension comprises the following steps: weighing compressed Fuzhuan tea, adding sterile water, mixing, shaking uniformly, and filtering tea residue to obtain Eurotium cristatum suspension.
Preferably, the preparation method of the eurotium cristatum spore suspension comprises the following steps: mixing the eurotium cristatum strain with sterile water to obtain eurotium cristatum spore suspension.
Preferably, the dilution is 50-fold of the bacterial suspension.
Compared with the prior art, the invention has the following beneficial effects:
the method for culturing eurotium cristatum by using different tea water is different from the traditional eurotium cristatum liquid production process, the method adopts the tea soup steamed at high temperature as the growth culture medium of the eurotium cristatum, the tea soup steamed at high temperature contains sugar, reducing sugar and uronic acid, the growth requirements of strains can be met, the tea soup is not limited to common dark green tea in the market, various tea systems are adopted for fermentation production of the eurotium cristatum, the unicity of the current production means of the eurotium cristatum and the uncontrollable quality improvement of the eurotium cristatum produced by the Fuzhuan tea through natural 'flowering' can be overcome, and the abundance of the production process is increased. Culturing in a solid tea soup culture medium at the temperature of 28-30 ℃ which is most suitable for the growth of eurotium cristatum for 8-10 days, and better observing the growth forms of the eurotium cristatum in different tea water solid culture media; in the liquid tea soup culture medium, the 7% v/v inoculation amount and the 40% v/v sample loading amount enable eurotium to grow more vigorously and more. The method is simple and easy to implement, convenient to control and suitable for industrial production, can be inoculated into other tea leaves with poor taste, such as ilex latifolia, and can improve the bitter taste and astringent taste of ilex latifolia, and the produced eurotium cristatum strain can be applied to the pharmaceutical industry, the food industry and other emerging industries. In addition, the method also solves the problem of single fermentation production of the past eurotium cristatum liquid, and the fermentation product eurotium cristatum strain and the secondary metabolite thereof can be further developed and researched by people, so that large-scale production can be realized.
Drawings
FIG. 1 is a drawing of loose tea and ground tea powder of different tea waters according to the present invention;
FIG. 2 is a graph showing the growth of different tea-like Eurotium cristatum strains starting on the third day of the solid fermentation according to the present invention; wherein, black tea (A group), oolong tea (B group), and yellow tea (C group);
FIG. 3 is a microscopic structure of Eurotium cristatum cultured with black tea of the present invention; wherein A is 100 times, B is 200 times, and C is 400 times;
FIG. 4 is a sequence diagram of the identification of Eurotium cristatum strain cultured from black tea of the present invention;
FIG. 5 is a liquid fermentation growth diagram of Eurotium cristatum of the present invention; wherein A is a black tea culture medium, B is an oolong tea culture medium, C is a yellow tea culture medium, D is a green tea culture medium, E is a black tea culture medium, and F is a white tea culture medium.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, shall fall within the protection scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the accompanying drawings:
the invention provides a method for culturing eurotium cristatum by using different tea water, which specifically comprises the following steps:
s1, preparing eurotium cristatum suspension: weighing a certain mass of compressed Fuzhuan tea, adding sterile water, mixing and shaking uniformly, and filtering tea residues with gauze to obtain the eurotium cristatum suspension. Inoculating the eurotium cristatum suspension on an improved CZG plate culture medium through aseptic operation, and culturing for 5-6 days at 28-30 ℃ to obtain eurotium cristatum strains;
wherein the mass ratio of the Fuzhuan tea to the sterile water is 1: 30, of a nitrogen-containing gas; the modified CZG plate medium was prepared from agar powder, sucrose, ferrous sulfate, magnesium sulfate, potassium chloride, dipotassium hydrogen phosphate, sodium nitrate and distilled water in a ratio of 2000: 3000: 1: 50: 50: 100: 200: 100000 mass ratio, natural pH value.
S2, preparing the powdered tea powder: preparing loose tea of different varieties with superior quality, drying the loose tea for 6 hours at 40-60 ℃, and crushing and sieving the loose tea by a crusher to obtain loose tea powder;
wherein the loose tea of different varieties comprises raw dark green tea, yellow tea, oolong tea, black tea and green tea; the power of the pulverizer is 1000W, and the pulverized material is sieved by a 80-mesh sieve;
s3, preparing different tea water: according to the weight percentage of the tea powder (g): weighing powdered tea powder and distilled water according to the proportion of 1:100 in distilled water (mL), steaming at the high temperature of 60-90 ℃ for 2h, and filtering the tea powder by using gauze to obtain different tea water;
s4, performing solid fermentation on eurotium cristatum of different tea leaves: adding agar powder into the different tea water obtained in the step S3 according to the proportion of 2%, sterilizing at 121 ℃ for 20min under natural pH value, and pouring the mixture while the mixture is hot to serve as a solid culture medium of eurotium cristatum for later use; diluting the bacterial suspension in the S1 by 50 times, inoculating the bacterial suspension on a solid tea soup culture medium, culturing for 8-10 days at 28-30 ℃, and observing the growth condition of the eurotium cristatum strain;
s5, fermenting different tea water eurotium cristatum liquid: taking different tea water obtained in S3 as a liquid culture medium of eurotium cristatum, sterilizing at a high temperature of 121 ℃ for 20min under a natural pH value, inoculating eurotium cristatum spore suspension into sterilized tea water with a sample loading amount of 40% v/v in an aseptic operation according to an inoculation amount of 7% v/v, culturing at 28 ℃ for 6d at 150r/min, and observing the liquid growth condition of the eurotium cristatum;
wherein, the eurotium cristatum spore suspension is obtained by uniformly mixing eurotium cristatum strains in S1 with sterile water.
Example 1
(1) Preparation of CZG Medium: weighing 20g of agar powder, 30g of sucrose, 0.01g of ferrous sulfate, 0.5g of magnesium sulfate, 0.5g of potassium chloride, 1g of dipotassium hydrogen phosphate and 2g of sodium nitrate into a beaker, adding 1000mL of distilled water and natural pH value, putting the beaker into a magnetic stirrer to be fully dissolved, sterilizing the beaker at the high temperature of 121 ℃ for 20min under the pressure of 0.1MPa in an autoclave, and subpackaging the beaker on a flat plate for standby when the beaker is hot.
(2) Preparing eurotium cristatum suspension: weighing 1g of compressed Fuzhuan tea, adding 30mL of sterile water, fully shaking to enable a yellow closed capsule shell on the surface of the tea to shake off in the sterile water, and filtering tea residues with gauze to obtain the Eurotium cristatum suspension.
(3) And (3) coating the eurotium cristatum suspension obtained in the step (2) on the CZG culture medium prepared in the step (1) on an ultra-clean workbench, and then putting the culture medium into an incubator for constant-temperature culture at 28 ℃ for 5d to obtain eurotium cristatum strains.
(4) Preparing the powdered tea powder: preparing tea samples (including Jingyang black tea, Anhui yellow tea, Fujian oolong tea, Hanzhong black tea, Hanzhong green tea and Fujian green tea) of different varieties with superior quality, drying at 60 ℃, crushing by a crusher, and sieving by a 80-mesh sieve to obtain powdered tea powder; fig. 1 shows different tea-like and powdered tea, wherein a1 is yellow tea, a2 is yellow tea powder, B1 is white tea, B2 is white tea powder, C1 is oolong tea, C2 is oolong tea powder, D1 is black tea, D2 is black tea powder, E1 is green tea, E2 is green tea powder, F1 is black tea, and F2 is black tea powder.
(5) Preparing different tea water: weighing 5g of different ground powdered tea powder in a beaker, adding distilled water to a constant volume of 500mL, and steaming at a high temperature of 90 ℃ for 2 h; filtering the tea residue with gauze to obtain different tea water for fermentation culture.
(6) Solid fermentation: measuring 150mL of tea water obtained in the step (4), adding 3g of agar powder, sterilizing at the high temperature of 121 ℃ for 20min under the pressure of 0.1MPa, pouring the tea water while the tea water is hot, cooling, performing aseptic operation, inoculating the tea water into the bacterial suspension diluted by 50 times in the step (2), then placing the tea water into an incubator for constant-temperature culture at the temperature of 28 ℃ for 9 days, observing the growth condition of the eurotium cristatum strain, wherein a graph 2 shows the growth graphs of the eurotium cristatum strain of black tea (A group), oolong tea (B group) and yellow tea (C group) starting on the third day of solid fermentation, the bacterial strain can be seen from the graphs, the bacterial strain can grow vigorously, the surface color of a culture medium, the height of the bacterial colony, the edge radiation degree of the bacterial colony and the texture of the bacterial colony are researched, the situation that the eurotium cristatum starts to grow from the third day of fermentation culture is found, the bacterial colony begins to grow and becomes large and dense along with the increase of time, starts to present a regular circle, the color of the bacterial colony is further deepened, and the surface is not smooth, the capsule shell has golden yellow granular shape, and the edge of the capsule shell spreads radially to the periphery. FIG. 3 is a microscopic structure diagram of 100, 200, 400 times of Eurotium cristatum cultured by black tea, under an optical microscope, the ascocarp is spherical or nearly spherical, and a large number of hyphae are separated around the ascocarp, the ascocarp contains a large number of ascomycetes, and can rupture and release ascomycetes in the middle and later growth period, and the ascospores can be released by the ascomycetes; and then identifying the strain of the eurotium cristatum cultured by the black tea, comparing the ITS zone sequence in an NCBI database, and displaying the identification result as shown in figure 4, wherein the strain is the eurotium cristatum.
(7) Liquid fermentation: and (3) taking a triangular flask with the volume of 500mL, adding 200mL of tea water obtained in the step (4), sterilizing at the high temperature of 121 ℃ for 20min under the condition of natural pH value and 0.1MPa, cooling, inoculating the Eurotium cristatum spore suspension in an aseptic operation with the inoculum size of 7%, placing the Eurotium cristatum spore suspension in a shaking table for culturing for 6d at the temperature of 28 ℃ and 150r/min, and observing the liquid growth condition of the Eurotium cristatum. Fig. 5 is a liquid fermentation growth diagram of eurotium cristatum, wherein a is a black tea culture medium, B is an oolong tea culture medium, C is a yellow tea culture medium, D is a green tea culture medium, E is a black tea culture medium, and F is a white tea culture medium, and the eurotium cristatum mycelium pellets subjected to fermentation culture by the black tea culture medium, the oolong tea culture medium, and the yellow tea culture medium are larger and denser than the green tea culture medium, the white tea culture medium, and the black tea culture medium.
Example 2
(1) Preparation of CZG Medium: weighing 20g of agar powder, 30g of sucrose, 0.01g of ferrous sulfate, 0.5g of magnesium sulfate, 0.5g of potassium chloride, 1g of dipotassium hydrogen phosphate and 2g of sodium nitrate into a beaker, adding 1000mL of distilled water and natural pH value, putting the beaker into a magnetic stirrer to be fully dissolved, sterilizing the beaker at the high temperature of 121 ℃ for 20min under the pressure of 0.1MPa in an autoclave, and subpackaging the beaker on a flat plate for standby when the beaker is hot.
(2) Preparing eurotium cristatum suspension: weighing 1g of compressed Fuzhuan tea, adding 30mL of sterile water, fully shaking to enable yellow cyst closure shells on the surface of the tea to shake off in the sterile water, and filtering tea residues with gauze to obtain the eurotium cristatum suspension.
(3) And (3) coating the eurotium cristatum suspension obtained in the step (2) on the CZG culture medium prepared in the step (1) on an ultra-clean workbench, and then putting the culture medium into an incubator for constant temperature culture at 29 ℃ for 6d to obtain eurotium cristatum strains.
(4) Preparing the powdered tea powder: preparing tea samples (including Jingyang black tea, Anhui yellow tea, Fujian oolong tea, Hanzhong black tea, Hanzhong green tea and Fujian green tea) of different varieties with superior quality, drying at 55 deg.C, pulverizing with a pulverizer, and sieving with a 80-mesh sieve to obtain powdered tea powder.
(5) Preparing different tea water: weighing 5g of different ground powdered tea powder in a beaker, adding distilled water to a constant volume of 500mL, and steaming at a high temperature of 60 ℃ for 2 h; filtering the tea residue with gauze to obtain different tea water for fermentation culture.
(6) Solid fermentation: weighing 150mL of tea water obtained in the step (4), adding 3g of agar powder, sterilizing at the high temperature of 121 ℃ under 0.1MPa for 20min, pouring the tea water into a plate while the tea water is hot, cooling, performing aseptic operation, inoculating the tea water into the bacterial suspension diluted by 50 times in the step (2), then placing the tea water into an incubator for constant temperature culture at 30 ℃ for 9 days, and performing strain identification on the eurotium cristatum cultured by the black tea.
(7) Liquid fermentation: and (3) taking a triangular flask with the volume of 500mL, adding 200mL of tea water obtained in the step (4), sterilizing at the high temperature of 121 ℃ for 20min under the condition of natural pH value and 0.1MPa, cooling, inoculating the Eurotium cristatum spore suspension in an aseptic operation with the inoculum size of 7%, placing the Eurotium cristatum spore suspension in a shaking table for culturing for 6d at the temperature of 28 ℃ and 150r/min, and observing the liquid growth condition of the Eurotium cristatum.
Example 3
(1) Preparation of CZG Medium: weighing 20g of agar powder, 30g of sucrose, 0.01g of ferrous sulfate, 0.5g of magnesium sulfate, 0.5g of potassium chloride, 1g of dipotassium hydrogen phosphate and 2g of sodium nitrate into a beaker, adding 1000mL of distilled water and natural pH value, putting the beaker into a magnetic stirrer to be fully dissolved, sterilizing the beaker at the high temperature of 121 ℃ for 20min under the pressure of 0.1MPa in an autoclave, and subpackaging the beaker on a flat plate for standby when the beaker is hot.
(2) Preparing eurotium cristatum suspension: weighing 1g of compressed Fuzhuan tea, adding 30mL of sterile water, fully shaking to enable yellow cyst closure shells on the surface of the tea to shake off in the sterile water, and filtering tea residues with gauze to obtain the eurotium cristatum suspension.
(3) And (3) coating the eurotium cristatum suspension obtained in the step (2) on the CZG culture medium prepared in the step (1) on an ultra-clean workbench, and then putting the culture medium into an incubator for constant temperature culture at 30 ℃ for 5d to obtain eurotium cristatum strains.
(4) Preparing the powdered tea powder: preparing tea samples (including Jingyang black tea, Anhui yellow tea, Fujian oolong tea, Hanzhong black tea, Hanzhong green tea and Fujian green tea) with different varieties with superior quality, drying at 40 deg.C, pulverizing with a pulverizer, and sieving with a 80-mesh sieve to obtain powdered tea powder.
(5) Preparing different tea water: weighing 5g of different ground powdered tea powder in a beaker, adding distilled water to a constant volume of 500mL, and steaming at a high temperature of 80 ℃ for 2 h; filtering the tea residue with gauze to obtain different tea water for fermentation culture.
(6) Solid fermentation: weighing 150mL of tea water obtained in the step (4), adding 3g of agar powder, sterilizing at the high temperature of 121 ℃ under 0.1MPa for 20min, pouring the tea water into a plate while the tea water is hot, cooling, performing aseptic operation, inoculating the tea water into the bacterial suspension diluted by 50 times in the step (2), then placing the tea water into an incubator for 10 days at the constant temperature of 29 ℃, and performing strain identification on the eurotium cristatum cultured by the black tea.
(7) Liquid fermentation: and (3) taking a triangular flask with the volume of 500mL, adding 200mL of tea water obtained in the step (4), sterilizing at the high temperature of 121 ℃ for 20min under the condition of natural pH value and 0.1MPa, cooling, inoculating the Eurotium cristatum spore suspension in an aseptic operation with the inoculum size of 7%, placing the Eurotium cristatum spore suspension in a shaking table for culturing for 6d at the temperature of 28 ℃ and 150r/min, and observing the liquid growth condition of the Eurotium cristatum.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (10)
1. A method for culturing Eurotium cristatum by using different tea water is characterized in that Eurotium cristatum suspension is diluted and inoculated on a solid tea soup culture medium, and cultured for 8-10 days at 28-30 ℃; the solid tea soup culture medium is prepared by adding agar powder into different tea water according to the proportion of 2 percent and sterilizing at high temperature under the condition of natural PH value;
or inoculating the eurotium cristatum spore suspension into a liquid tea soup culture medium with the sample loading of 40% v/v according to the inoculation amount of 7% v/v, and culturing at 28 ℃ and 150r/min for 6 d; the liquid tea soup culture medium is prepared by sterilizing different tea water at natural pH for 20min at high temperature.
2. The method for culturing eurotium cristatum in different tea water as claimed in claim 1, wherein the preparation method of different tea water comprises: drying, crushing and sieving different kinds of loose tea to obtain loose tea powder, and steaming and boiling the loose tea powder and distilled water at high temperature and filtering to obtain different tea water.
3. The method of claim 2, wherein the different loose teas comprise raw dark green tea, yellow tea, oolong tea, black tea, and green tea.
4. The method for culturing eurotium cristatum with different tea water as claimed in claim 2, wherein the drying is 40-60 ℃.
5. The method for culturing eurotium cristatum with different tea water as claimed in claim 2, wherein the powdered tea is mixed with distilled water at a ratio of 1g:100 mL.
6. The method for culturing eurotium cristatum with different tea water as claimed in claim 2, wherein the high temperature cooking is 60-90 ℃.
7. The method for culturing eurotium cristatum in different tea waters as claimed in claim 2, wherein the pulverizing and sieving is carried out by pulverizing with a pulverizer having a power of 1000W and sieving with a 80 mesh sieve.
8. The method for culturing eurotium cristatum by using different tea water as claimed in claim 1, wherein the preparation method of the eurotium cristatum suspension comprises: weighing compressed Fuzhuan tea, adding sterile water, mixing, shaking uniformly, and filtering tea residue to obtain Eurotium cristatum suspension.
9. The method for culturing eurotium cristatum in different tea water as claimed in claim 1, wherein the preparation method of the eurotium cristatum spore suspension comprises: mixing the eurotium cristatum strain with sterile water to obtain eurotium cristatum spore suspension.
10. The method for culturing Eurotium cristatum with different tea water as claimed in claim 1, wherein the dilution is 50 times that of the bacterial suspension.
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| CN110613032A (en) * | 2019-08-15 | 2019-12-27 | 福州大学 | Method for establishing basidiomycete fermentation system of novel aroma-enhanced tea beverage |
| CN113439791A (en) * | 2021-07-12 | 2021-09-28 | 广西民族大学 | Fuzhuan tea making process |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114108151A (en) * | 2021-12-03 | 2022-03-01 | 王文俊 | Antistatic soft wool fabric and preparation method thereof |
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